1. Field of the Invention
This invention relates to an Italian rye grass into which endophytic fungi are introduced and a method of introducing endophytic fungi into Italian rye grass. Italian rye grass is a member of the genus Lolium of the tribe Poeae, which is in the sub-family of grasses known as Pooideae.
2. Description of the Related Art
Lolium in the genus Poa, which belongs to the sub-family of grasses known as Pooideae, includes Italian rye grass (Lolium multiflorum), perennial rye grass (Lolium perenne), and dock wheat (Lolium temuletum). Italian rye grass is also referred to by the Japanese name of rat wheat. This plant not only grows naturally, but is also widely cultivated artificially as meadow grass and is very useful for livestock.
Italian rye grass has many uses, and as it is used over a large area, it is easily prone to disease and insects. There is a large amount of damage due to Bluegrass webworm, and this can be so serious that whole meadows can disappear overnight in locations where the chemical spraying of insect larvae, which have just hatched, has not reached.
Conventional methods of cultivating and growing grasses include the artificial crossing method, selection method, mutation method, cell fusion method and gene insertion method. Due to recent progress in biotechnology, the cultivation period which previously required 10 years or more, has been reduced to several years. As regards genetic insertion, several techniques exist such as a method using agrobacterium, the electroporation method and the particle gun method, and they are now being applied to a large variety of crops.
However, in the case of grasses, it has been pointed out that this genetic insertion is extremely inefficient. For example, it is difficult to infect grasses using the agrobacterium method, so genetic insertion is very difficult. As regards the electroporation method, a regeneration system has to be developed from the protoplast of the grass, and even if such regeneration is possible, the characteristics of the plant may suffer damage due to growth mutations.
Concerning the particle gun method, since genes are introduced randomly into the plant organism or culture, the plant obtained frequently becomes a chimera.
In the case of Lolium including Italian rye grass, cell culture techniques such as cell fusion or genetic insertion require complex operating procedures and they are not very efficient. Thus, there are very few examples where they have successfully been applied efficiently, and there are very few instances where they have been successfully developed on a practical level.
However, there are some wild plants in nature in which filamentous internal fungi, i.e. endophytes, live together with the plant. They grow well in plant tissue, and particularly well in the gaps between cells, i.e. the intercellular spaces.
These endophytes, or symbiotic filamentous fungi, not only have no adverse effect on the host plant but in fact provide it with useful substances, and contribute to help it withstanding environmental stresses.
Enhancement of plant properties by endophytes is known from the literature, e.g. insect resistance (Siegel et al, 1987, Ann. Rev. Phytopathol. 25: 293-3 15), disease resistance (Gwinn and Gavin, 1992, Plant Disease 76: 911-914), environmental stress (drought, etc.) resistance (Arachevalta et al, 1989, Agron. J. 81: 83-90), and growth enhancement (Latch et al, 1985, N.Z.J. Agric. Res. 28: 165-168). It is particularly well-known that, in perennial rye grass infected with endophytes, these endophytes improve insect resistance due to the repelling substances and alkaloids they produce.
Latch et al in New Zealand are searching for an endophyte known as an endosafe which has a low toxicity to livestock and excellent insect resistance by collecting and studying endophytes in perennial rye grass.
However, many of the plants in which these endophytes live have little utility, so it is necessary to introduce them into useful grasses. In this regard, attempts have already been made to introduce endophytes into perennial rye grass which is an important pasture grass. The techniques used may be broadly distinguished as artificial crossing and artificial inoculation.
In artificial crossing, useful characteristics are introduced by pollen using a plant infected by an endophyte as mer. However, in the conventional method, there were limitations on the species and strains which could be crossed with one another. In artificial inoculation, plants or culture tissues are inoculated with endophytes that have been isolated and cultivated.
The inoculation method is capable of introducing a wider range of types, but due to problems of technique regarding cultivation of endophytes, inoculation conditions and conditions of the plant itself, it is limited to perennial rye grass. To increase the infection rate, a method has been reported where callus is used as the plant tissue which is inoculated. However according to this method, it is necessary to develop a plant regenerating system from the callus, hence the method was still limited to perennial rye grass.
The conventional cell cultivation method involves a troublesome procedure and requires considerable training. Its practical application was moreover difficult since culture mutations caused by transformation or cell fusion had an effect on the characteristics being introduced or on other traits.
In the genetic insertion method, it was not possible to introduce specific characteristics if it was not known which genes had an effect on the characteristics and type of plant.
Characteristics related to complex factors such as environmental stress could not be introduced by techniques such as genetic insertion. Moreover, plants grown by cell culture techniques were often found to exhibit decreased seed fertility. In the case of grasses this led to a decline of yield and was therefore fatal.
In view of this situation, growth techniques or improvement of characteristics using endophytes is a totally new approach to solving the above problems.
When endophytes are introduced into plants by artificial inoculation on the other hand, the technique is limited to perennial rye grass due to problems in searching for endophytes and cultivation systems, and it has never been applied to other useful grasses such as Italian rye grass. In callus inoculation, it is essential to develop a regenerating system of the plant into which the endophyte is to be introduced. Moreover, inoculation conditions had not been developed to increase the rate of infection.
At present, useful natural endophytes have been found only in perennial rye grass, tall fescue and meadow fescue resulting in a major limitation to their introductions. In particular, the host plants of these endophytes are foreign types, and no endophytes derived from domestic plants had yet been found that were adapted to Japanese environmental conditions.
It is therefore an object of this invention to provide an Italian rye grass into which an endophyte is artificially introduced, and a method of artificially introducing an endophyte into Italian rye grass not infected with endophyte.
Another object of this invention is to provide an Italian rye grass with excellent characteristics, and in particular, excellent insect resistance and disease resistance.
According to one aspect of this invention, there is provided an Italian rye grass produced by artificially introducing filamentous endophytic fungi into plants not containing filamentous endophytic fungi. Here, Italian rye grass includes plants having the scientific name Lolium multiflorum, and also includes hybrids of Italian rye grass.
The endophytic fungi may be one or both of Neotyphodium and Gliocladium. The endophytic fungi may produce an insect-resistant alkaloid. The Italian rye grass may also be used as turf. The endophytic fungi may be any of FERM P-14798, 15029, 15030, 15862, 16319, 16320, 16327, 14797, 16103, 17372, 14799, 14800, 16321, 16322, 16323, 16324, 16325, 16326, 16328, 16329 or 17351 deposited at the Japanese National Institute of Bioscience and Human Technology.
The invention relating to a method of introducing endophytic fungi into an Italian rye grass, is a method comprising:
a step for isolating filamentous endophytic fungi living naturally in a symbiotic relationship with a plant, and artificially growing the fungi,
a step for artificially inoculating the grass with the artificially grown endophytic fungi, and
a step for infecting the grass with the artificially inoculated endophytic fungi.
The endophytic fungi may be artificially inoculated by inoculating conidiospores of said endophytic fungi. The characteristics of the isolated endophytic fungi may be examined, and selected endophytic fungi may be inoculated. The characteristics may be any of insect resistance, disease resistance, environmental stress resistance or growth enhancement.
The endophytic fungi may be one or both of Neotyphodium and Gliocladium. The said endophytic fungi may produce an insect-resisting alkaloid. The endophytic fungi are any of FERM P-14798, 15029, 15030, 15862, 16319, 16320, 16327, 14797, 16103, 17372, 14799, 14800, 16321, 16322, 16323, 16324, 16325, 16326, 16328, 16329 or 17351 deposited at the Japanese National Institute of Bioscience and Human Technology.
As described above, this invention relates to Italian rye grasses produced by artificially introducing filamentous endophytic fungi, i.e., endophytes, into Italian rye grasses not containing endophytes, and to a method of artificially introducing endophytes into Italian rye grasses.
Therefore, according to this invention, an endophytic fungus, i.e., an endophyte, is introduced into an Italian rye grass, and by making the endophytic fungus live symbiotically in the Italian rye grass, it is possible to confer at least insect resistance and disease resistance. Therefore, the characteristics of the Italian rye grass are improved, and an Italian rye grass having excellent properties can be obtained.
The above and other objects, features and advantages of this invention will be apparent from the following descriptions of practical mode and examples.
The method of introducing endophytes into an Italian rye grass according to another aspect of this invention will now be described in still more detail practically.
Stage 1 Detection of presence or absence of endophyte and isolation of same
(1) Detection of endophyte infection
An epidermis of leaf with its sheath is removed from a plant collected in a search, the leaf is peeled and stained with aniline blue solution, and any endophyte in the tissue is detected by examination with an optical microscope.
(2) Isolation and culture of endophyte
After sterilizing plant sections confirmed to contain endophyte, the section is transplanted to an endophyte isolation culture and cultured for several months.
(3) Endophyte classification
Isolated endophyte is classified according to the host, or cultured by varying environmental conditions using the flat plate culture method and classified according to its morphology. Alternatively, a liquid culture is performed and the endophyte is classified according to its morphology, or a slide culture is performed and the endophyte is classified according to its morphology.
Stage 2 Alkaloid analysis
Alkaloid produced either by the fungus alone or when living on the plant is analyzed and examined in particular for insect resistance. Analysis for disease resistance, environmental stress resistance and growth enhancement may be made at the same time.
Stage 3 Introduction of endophyte
The isolated endophyte is artificially introduced into the desired or target Italian rye grass. The endophyte may be introduced by directly inoculating the plant with it, alternatively non-differentiated cells such as callus can be inoculated and the plant is regenerated from the callus. An appropriate method should be chosen according to the type of plant in which it is desired to introduce the endophyte.
Stage 4 Confirmation of endophyte presence
An explant from a plant into which endophyte has been introduced is stained with a dye solution, observed with an optical microscope, and the presence of the endophyte or infection by it is detected using the enzyme immunoassay method.
Stage 5 Examination of plants into which endophyte has been introduced
(1) Resistance to pests
Using plants into which endophyte had been introduced and plants not containing endophyte, larvae of pests are grown, and a survey of pest damage is carried out artificially.
(2) Resistance to disease
Using Italian rye grass into which endophyte has been introduced, and the same Italian rye grass not containing endophyte, resistance to disease is compared by artificially inoculating the two types of plants with pathogenic fungi and examining the extent of disease.
(3) Tests with later generations of plants
Seeds containing endophyte are collected, germinated, and after confirming that the endophyte was present, the aforesaid tests are performed.